The development of neurobiological models has contributed significantly to the understanding of brain function. An important model of brain function at the cellular level is the synapse because the synapse mediates neuronal interconnections. By virtue of involvement in chemical neurotransmission, receptor proteins are critical to synapses. The neuromuscular synapse and its receptor, the nicotinic acetylcholine receptor (nAChR) have been used to model the relationship between receptors and synaptic function. Muscle cells express nAChR as they develop into myotubes. Innervation localizes nAChR to the synapse and alters nAChR metabolism. Denervation may reverse the effects of innervation. Transcriptional regulation is a major control point of eukaryotic gene expression. Changes in nAChR expression are likely to be mediated, at least in part, by regulation of transcription. Differentiation of a nAChR expressing mouse cell line is accompanied by an increase in mRNA for the Alpha nAChR subunit. Denervation of mouse muscle is accompanied by a large increase in Alpha subunit mRNA. Although altered post transcriptional processing may play a role in these increases, it is necessary to consider transcriptional regulation of the subunit genes as a cause for the increase in mRNA prior to considering the regulation of post transcriptional processing. As a first step to studying nAChR transcriptional regulation, I propose to characterize the genes for the nAChR subunits. Homologous mouse subunit cDNA clones will be employed to screen mouse Lambda genomic libraries. Mouse Alpha and Delta cDNA's are available; Beta and Gamma will be prepared. Genomic clones will be characterized by restriction enzyme mapping and nucleotide sequencing. The analysis of gene structure will also consider gene linkage. To study transcriptional regulation it is necessary to define the transcriptional unit. Initial efforts will focus on the site transcription initiation. S1 nuclease protection and primer extension of subunit mRNA will define the 5' end of transcription.
